The present invention relates to an adsorbent for removing a chemokine in body fluid, a method for removing a chemokine in body fluid by means of the adsorbent, and an adsorber for removing a chemokine from body fluid.
An immunocompetent cell produces various kinds of active substances when causing immune response. One portion thereof is a proteinous substance called a cytokine and plays a greatly important role as a biophylactic factor which is closely related to various kinds of antigen-specific response and/or non-specific inflammatory response. Essentially, a cytokine is necessary and indispensable for maintaining biological homeostasis and is produced excessively in pathological conditions such as inflammation and the like, relating to the formation and the prolongation of inflammation and the like.
Among the cytokines, especially, ones having chemotaxis are generically named chemokines. Chemotaxis is also referred to as chemotropism, and means tropism caused by difference in concentration of a chemical entity. It is known that the substances referred to as chemokines form one family for their structural characteristics.
Chemokines are characterized in that they exist mainly as proteins having a molecular weight of from about 6,000 to about 10,000. Depending on kinds of chemokines, however, there exist chemokines which form dimers or tetramers in fluid, and chemokines having a molecular weight more than 10,000 because of O-glycosylation. Also, chemokines are classified into the following two subfamilies according to their structural characteristics. One is CXC subfamily, and the other is CC subfamily. As shown in the Review xe2x80x9cCC CHEMOKINES IN ALLERGIC INFLAMMATIONxe2x80x9d, Immunology Today, 15, 127-133, 1994 by M. Baggiolini et al., a chemokine has four cysteine residues (hereinafter a cysteine residue is referred to as C) in a position firmly conserved in its molecule. When such four Cs are referred to as C1, C2, C3 and C4 in that order from N-terminus, chemokines are classified into CXC subfamily wherein one optional amino acid (hereinafter referred to as X) exists between C1 and C2, and CC subfamily wherein no amino acid exists between C1 and C2. Further, it is shown that chemokines in each subfamily have relatively high homology in a sequence of amino acids other than Cs (e.g., the report by Chihara, Clinical Immunology, 27 [Suppl. 16], 162-171, 1995).
It has been thought that CXC subfamily acts mainly on a neutrophil among leukocytes, while CC subfamily acts mainly on a monocyte, an eosinophil, a basophil and a lymphocyte among leukocytes. Recently, however, it has been suggested that they exert their effects on various kinds of cells as well as the above-mentioned leukocytes. For instance, it is known that interleukin-8 (IL-8) shows physiological activities on a lymphocyte, a basophil, an eosinophil, an epidermal keratinocyte, a melanomatous cell, a fibroblast and an endothelial cell as well as a neutrophilic, although interleukin-8 is an interleukin having chemotaxis among interleukins and is a chemokine classified into CXC subfamily (Matsushima, Clinical Immunology, 27 [Suppl. 16], 147-154, 1995).
Further, it is known that, on e.g., a human monocyte, there exist not only receptors specific to each of monocyte chemoattractant protein-1 (hereinafter referred to as MCP-1) and macrophage inflammatory protein-1 (hereinafter referred to as MIP-1) which are chemokines classified into CC subfamily, but also a common receptor specific to three kinds of chemokines classified into CC subfamily, i.e., MCP-1, MIP-1 and RANTES, (Regulated upon Activation in Normal T cells Expressed and Secreted) (Matsushima, Clinical Immunology, 27 [Suppl. 16], 147-154, 1995). This finding suggests that there exist chemokines in one subfamily which exert the same phisiological activity through the same receptor.
Once a living body has stress or infection from the out side, inflammation is caused as a biophylactic response, and there arises infiltration of leukocytes into an inflammatory site. Such infiltration of leukocytes into an inflammatory site is caused by leukocyte chemotactic factor produced at the inflammatory site. It is known that a chemokine plays a role as a causative factor of the infiltration of leukocytes. In fact, it has been demonstrated that administration of an antibody against interleukin-8 (anti-IL-8-antibody) being one of chemokines blocks infiltration of neutrophilics at a inflammatory site and inhibits a disorder of organ accompanied with acute inflammation in a model of a rabbit with acute inflammation (Sekido et al., Nature, 365, 654-657, 1993).
Furthermore, recently it has been reported that a network of cytokines is activated by overproduction of various cytokines, and induction and activation of neutrophils are caused by overproduction of chemokines due to the activation of the network of cytokines, in pathological conditions included in a conception of systemic inflammatory response syndrome (SIRS) (Endo et al., Intensive and Critical Care Medicine, 4, 1357-1365, 1992). It is suggested that thereby systemic inflammatory response progresses, and shock, a tissue disorder and pluriorganic insufficiency are caused, and then death may come.
It is suggested that at a pathologic site of allergic inflammation, various inflammatory cells such as a lymphocyte and an eosinophil infiltrate according to action of chemokines such as RANTES, platelet factor-4 (hereinafter referred to as PF-4) and macrophage inflammatory protein-1xcex1 (hereinafter referred to as MIP-1xcex1), as key substances.
Also, for instance, in case of carrying out blood extracorporeal circulation such as dialysis therapy, the possibility has been suggested that chemokines are overproduced by stimulation to an immunocompetent cell by means of contact with an artificial material, an irritant represented by microbial endotoxin in dialysis, various irritant factors existing in blood or tissue, and the like. For instance, in dialysis amyloidosis or carpal tunnel syndrome which is a complication accompanied with a long-term dialysis therapy, the possibility has been suggested that MCP-1 or MIP-1xcex1 is overproduced and relates to formation of pathological conditions (Inoue et al., Nephrology Dialysis Transplantation, 10, 2077-2082, 1995).
Further, an abnormally higher concentration of interleukin-8 being one of chemokines has been detected at an inflammatory site or in peripheral blood of patients with diseases such as gouty arthritis, psoriasis, contact dermatitis, idiopathic fibroid lung, adult respiratory distress syndrome, inflammatory bowel disease, immune angiitis, urinary tract infection cardiac infarction, asthma, respiratory tract infection, perinatal infectious disease and rejection in organ transplantation, than that of a normal human (Menekiyakuri, 12, No. 1, 15-21, 1994).
Also, there abnormally appear interleukin-8, RANTES, MCP-1, MIP-1xcex1 and macrophage inflammatory protein-1xcex2 (hereinafter referred to as MIP-1xcex2) in rheumatoid arthritis (RA); MCP-1, MIP-1xcex1 and MIP-1xcex2 in crescentic glomerulonephritis; interleukin-8 and MCP-1 in chronic glomerular nephritis; and MCP-1 in lupus nephritis. It is suggested that the chemokines concern formation of pathological conditions of the above-mentioned diseases.
Until now, there is no report as to a method for removing such chemokines which have various functions in body fluid. There is disclosed only a method for purifying blood with an adsorbent for removing an endotoxin and/or a cytokine caused by the endotoxin, which comprises a porous carrier having a cationic functional group on the surface (Japanese Unexamined Patent Publication No. 312017/1994). However, there is described neither measurement of the cytokine nor adsorption of the cytokine in Example thereof.
It is also considered that inhibition of chemokine""s action, so-called anti-chemokine therapy is applied by administering an antibody against the chemokine or a substance which inhibits binding of the chemokine to a receptor thereof. However, it is necessary to prepare and administer an antibody against each chemokine in order to inhibit such action by administration of the antibody or the like, because it is suggested that many kinds of chemokines abnormally appear in pathologic conditions accompanied with chronic inflammation such as the above-mentioned rheumatoid arthritis. Further, an antibody or the like to be administered must not exert bad influence upon a human body, and it is considered that development thereof requires long term and a great cost. Therefore, it is hard to say that such therapy is suitable one.
In order to solve the above problems, an object of the present invention is to provide an adsorbent which can efficiently adsorb and remove various chemokines present in body fluid.
A further object of the present invention is to provide a method for removing the chemokines by means of the above-mentioned adsorbent.
A still further object of the present invention is to provide an adsorber for removing the chemokines with the above-mentioned adsorbent.
These and the other objects of the present invention will become apparent from the following description.
By investigating literatures, it became clear hat there were many chemokines having at least 7 of isoelectric point, namely having a positive charge in a physiological condition, while there were several chemokines having less than 7 of isoelectric point, namely a negative charge in a physiological condition. As a result of the continuous effort of the present inventors with respect to an adsorbent having ability to efficiently adsorb and remove chemokines with various charges existing in body fluid, it has been found that a solid material having an anionic functional group, particularly a styrene-divinylbenzene copolymer having a sulfonic acid group, strongly adsorbes a chemokine regardless of the isoelectric point thereof, in case where the solid material is brought into contact with the chemokine. Consequently the present invention has been accomplished.
The present applicant has previously filed the patent applications concerning the adsorbent for removing interleukins, which comprises a solid material having an anionic functional group (Japanese Patent Application No. 226906/1994 (National Republication of PCT Application No. PCT/JP95/01859 (WO 96/09115)) and Japanese Patent Application No. 229298/1995 (Japanese Unexamined patent publication No. 281101/1996)). Therefore, among interleukins, interleukin-8 having chemotaxis, which is a chemokine classified into CXC subfamily and has 8.6 of the isoelectric point, is excepted from the chemokine of the present invention.
In accordance with the present invention, there is provided (1) an adsorbent for removing a chemokine except interleukin-8 in body fluid, which comprises a solid material having an anionic functional group.
Further, there is provided (2) the adsorbent of the above-mentioned item (1), wherein the anionic functional group comprises at least one kind of functional group selected from the group consisting of sulfuric ester group, sulfonic acid group, carboxyl group and phosphoric ester group.
Further, there is provided (3) the adsorbent of the above-mentioned item (1) or item (2), wherein the solid material having an anionic functional group is a solid material onto which a polyanionic compound having plural anionic functional groups in its molecule is immobilized.
Further, there is provided (4) the adsorbent of the above-mentioned item (3), wherein the polyanionic compound is a synthetic polyanionic compound and/or a synthetic acidic polysaccharide.
Further, there is provided (5) the adsorbent of the above-mentioned item (1), wherein the solid material having an anionic functional group is a styrene-divinylbenzene copolymer having a sulfonic acid group.
Further, there is provided (6) the adsorbent of the above-mentioned item (5), wherein ion-exchange capacity of the styrene-divinylbenzene copolymer having a sulfonic acid group is from 0.01 meq/ml to 5 meq/ml.
Further, there is provided (7) a method for removing a chemokine except interleukin-8, which comprises by bringing the adsorbent of the above-mentioned item (1) into contact with body fluid containing the chemokine.
Further, there is provided (8) the method of the above-mentioned item (7), wherein the adsorbent for removing the chemokine comprises a styrene-divinylbenzene copolymer having a sulfonic acid group.
Further, there is provided (9) the method of the above-mentioned item (8), wherein ion-exchange capacity of the styrene-divinylbenzene copolymer having a sulfonic acid group is from 0.01 meq/ml to 5 meq/ml.
Further, there is provided (10) the method of the above-mentioned item (8), wherein a vessel which has an inlet and an outlet for fluid contains the adsorbent for removing a chemokine in the vessel.
Further, there is provided (11) an adsorber for removing a chemokine except interleukin-8, wherein a vessel which has an inlet and an outlet for fluid and is equipped with a means to prevent the adsorbent of the above-mentioned item (1) from flowing to the outside of the vessel, is charged with the adsorbent of the above-mentioned item (1).
Further, there is provided (12) the adsorber of the above-mentioned item (11), wherein the adsorbent for removing the chemokine comprises a styrene-divinylbenzene copolymer having a sulfonic acid group.
A variety of chemokines except interleukin-8 existing in body fluid can be efficiently adsorbed and removed by means of an adsorbent according to the present invention, which comprises a solid material having an anionic functional group. The present invention can provide an effective method for suppressing the effect of chemokines in various diseases caused by the chemokines.